RT Journal Article
SR Electronic
T1 Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 393231
DO 10.1101/393231
A1 Channabasavaiah Gurumurthy
A1 Rolen Quadros
A1 John Adams, Jr
A1 Pilar Alcaide
A1 Shinya Ayabe
A1 Johnathan Ballard
A1 Surinder K. Batra
A1 Marie-Claude Beauchamp
A1 Kathleen A Becker
A1 Guillaume Bernas
A1 David Brough
A1 Francisco Carrillo-Salinas
A1 Ruby Dawson
A1 Victoria DeMambro
A1 Jinke D’Hont
A1 Katharine Dibb
A1 James D. Eudy
A1 Lin Gan
A1 Jing Gao
A1 Amy Gonzales
A1 Anyonya Guntur
A1 Huiping Guo
A1 Donald W. Harms
A1 Anne Harrington
A1 Kathryn E. Hentges
A1 Neil Humphreys
A1 Shiho Imai
A1 Hideshi Ishii
A1 Mizuho Iwama
A1 Eric Jonasch
A1 Michelle Karolak
A1 Bernard Keavney
A1 Nay-Chi Khin
A1 Masamitsu Konno
A1 Yuko Kotani
A1 Yayoi Kunihiro
A1 Imayavaramban Lakshmanan
A1 Catherine Larochelle
A1 Catherine B. Lawrence
A1 Lin Li
A1 Volkhard Lindner
A1 Xian-De Liu
A1 Gloria Lopez-Castejon
A1 Andrew Loudon
A1 Jenna Lowe
A1 Loydie Jerome-Majeweska
A1 Taiji Matsusaka
A1 Hiromi Miura
A1 Yoshiki Miyasaka
A1 Benjamin Morpurgo
A1 Katherine Motyl
A1 Yo-ichi Nabeshima
A1 Koji Nakade
A1 Toshiaki Nakashiba
A1 Kenichi Nakashima
A1 Yuichi Obata
A1 Sanae Ogiwara
A1 Mariette Ouellet
A1 Leif Oxburgh
A1 Sandra Piltz
A1 Ilka Pinz
A1 Moorthy P. Ponnusamy
A1 David Ray
A1 Ronald J. Redder
A1 Clifford J Rosen
A1 Nikki Ross
A1 Mark T. Ruhe
A1 Larisa Ryzhova
A1 Ane M. Salvador
A1 Radislav Sedlacek
A1 Karan Sharma
A1 Chad Smith
A1 Katrien Staes
A1 Lora Starrs
A1 Fumihiro Sugiyama
A1 Satoru Takahashi
A1 Tomohiro Tanaka
A1 Andrew Trafford
A1 Yoshihiro Uno
A1 Leen Vanhoutte
A1 Frederique Vanrockeghem
A1 Brandon J. Willis
A1 Christian S. Wright
A1 Yuko Yamauchi
A1 Xin Yi
A1 Kazuto Yoshimi
A1 Xuesong Zhang
A1 Yu Zhang
A1 Masato Ohtsuka
A1 Satyabrata Das
A1 Daniel J. Garry
A1 Tino Hochepied
A1 Paul Thomas
A1 Jan Parker-Thornburg
A1 Antony D Adamson
A1 Atsushi Yoshiki
A1 Jean-Francois Schmouth
A1 Andrei Golovko
A1 William R. Thompson
A1 KC. Kent Lloyd
A1 Joshua A. Wood
A1 Mitra Cowan
A1 Tomoji Mashimo
A1 Seiya Mizuno
A1 Hao Zhu
A1 Petr Kasparek
A1 Lucy Liaw
A1 Joseph M. Miano
A1 Gaetan Burgio
YR 2018
UL http://biorxiv.org/content/early/2018/09/01/393231.abstract
AB CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the method’s success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA–2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.